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. 1985 Oct 1;101(4):1463–1472. doi: 10.1083/jcb.101.4.1463

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage

PMCID: PMC2113903  PMID: 3930510

Abstract

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.

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Selected References

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  1. Allen R. D., Zacharski L. R., Widirstky S. T., Rosenstein R., Zaitlin L. M., Burgess D. R. Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy. J Cell Biol. 1979 Oct;83(1):126–142. doi: 10.1083/jcb.83.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Behnke O., Zelander T. Filamentous substructure of microtubules of the marginal bundle of mammalian blood platelets. J Ultrastruct Res. 1967 Jul;19(1):147–165. doi: 10.1016/s0022-5320(67)80065-0. [DOI] [PubMed] [Google Scholar]
  3. Boyles J., Bainton D. F. Changing patterns of plasma membrane-associated filaments during the initial phases of polymorphonuclear leukocyte adherence. J Cell Biol. 1979 Aug;82(2):347–368. doi: 10.1083/jcb.82.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carlsson L., Markey F., Blikstad I., Persson T., Lindberg U. Reorganization of actin in platelets stimulated by thrombin as measured by the DNase I inhibition assay. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6376–6380. doi: 10.1073/pnas.76.12.6376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carroll R. C., Butler R. G., Morris P. A., Gerrard J. M. Separable assembly of platelet pseudopodal and contractile cytoskeletons. Cell. 1982 Sep;30(2):385–393. doi: 10.1016/0092-8674(82)90236-7. [DOI] [PubMed] [Google Scholar]
  6. Casella J. F., Flanagan M. D., Lin S. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature. 1981 Sep 24;293(5830):302–305. doi: 10.1038/293302a0. [DOI] [PubMed] [Google Scholar]
  7. Daniel J. L., Holmsen H., Adelstein R. S. Thrombin-stimulated myosin phosphorylation in intact platelets and its possible involvement secretion. Thromb Haemost. 1977 Dec 15;38(4):984–989. [PubMed] [Google Scholar]
  8. Davies G. E., Palek J. The state of actin polymerization in tetracaine-treated platelets. Thromb Haemost. 1982 Oct 29;48(2):153–155. [PubMed] [Google Scholar]
  9. Debus E., Weber K., Osborn M. The cytoskeleton of blood platelets viewed by immunofluorescence microscopy. Eur J Cell Biol. 1981 Apr;24(1):45–52. [PubMed] [Google Scholar]
  10. Fox J. E., Boyles J. K., Reynolds C. C., Phillips D. R. Actin filament content and organization in unstimulated platelets. J Cell Biol. 1984 Jun;98(6):1985–1991. doi: 10.1083/jcb.98.6.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fox J. E., Dockter M. E., Phillips D. R. An improved method for determining the actin filament content of nonmuscle cells by the DNase I inhibition assay. Anal Biochem. 1981 Oct;117(1):170–177. doi: 10.1016/0003-2697(81)90707-7. [DOI] [PubMed] [Google Scholar]
  12. Fox J. E., Phillips D. R. Inhibition of actin polymerization in blood platelets by cytochalasins. Nature. 1981 Aug 13;292(5824):650–652. doi: 10.1038/292650a0. [DOI] [PubMed] [Google Scholar]
  13. Fox J. E., Phillips D. R. Polymerization and organization of actin filaments within platelets. Semin Hematol. 1983 Oct;20(4):243–260. [PubMed] [Google Scholar]
  14. Fox J. E., Phillips D. R. Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets. J Biol Chem. 1982 Apr 25;257(8):4120–4126. [PubMed] [Google Scholar]
  15. Gear A. R. Rapid platelet morphological changes visualized by scanning-electron microscopy: kinetics derived from a quenched-flow approach. Br J Haematol. 1984 Mar;56(3):387–398. doi: 10.1111/j.1365-2141.1984.tb03969.x. [DOI] [PubMed] [Google Scholar]
  16. Gonnella P. A., Nachmias V. T. Platelet activation and microfilament bundling. J Cell Biol. 1981 Apr;89(1):146–151. doi: 10.1083/jcb.89.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harris H. E., Weeds A. G. Platelet actin: sub-cellular distribution and association with profilin. FEBS Lett. 1978 Jun 1;90(1):84–88. doi: 10.1016/0014-5793(78)80303-2. [DOI] [PubMed] [Google Scholar]
  18. Jennings L. K., Fox J. E., Edwards H. H., Phillips D. R. Changes in the cytoskeletal structure of human platelets following thrombin activation. J Biol Chem. 1981 Jul 10;256(13):6927–6932. [PubMed] [Google Scholar]
  19. Lewis J. C., White M. S., Prater T., Porter K. R., Steele R. J. Three-dimensional organization of the platelet cytoskeleton during adhesion in vitro: observations on human and nonhuman primate cells. Cell Motil. 1983;3(5-6):589–608. doi: 10.1002/cm.970030525. [DOI] [PubMed] [Google Scholar]
  20. Lyons R. M., Stanford N., Majerus P. W. Thrombin-induced protein phosphorylation in human platelets. J Clin Invest. 1975 Oct;56(4):924–936. doi: 10.1172/JCI108172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Markey F., Persson T., Lindberg U. Characterization of platelet extracts before and after stimulation with respect to the possible role of profilactin as microfilament precursor. Cell. 1981 Jan;23(1):145–153. doi: 10.1016/0092-8674(81)90279-8. [DOI] [PubMed] [Google Scholar]
  22. Mattson J. C., Zuiches C. A. Elucidation of the platelet cytoskeleton. Ann N Y Acad Sci. 1981;370:11–21. doi: 10.1111/j.1749-6632.1981.tb29716.x. [DOI] [PubMed] [Google Scholar]
  23. Maupin-Szamier P., Pollard T. D. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978 Jun;77(3):837–852. doi: 10.1083/jcb.77.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nachmias V. T. Cytoskeleton of human platelets at rest and after spreading. J Cell Biol. 1980 Sep;86(3):795–802. doi: 10.1083/jcb.86.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nachmias V., Sullender J., Asch A. Shape and cytoplasmic filaments in control and lidocaine-treated human platelets. Blood. 1977 Jul;50(1):39–53. [PubMed] [Google Scholar]
  26. Pribluda V., Rotman A. Dynamics of membrane-cytoskeleton interactions in activated blood platelets. Biochemistry. 1982 Jun 8;21(12):2825–2832. doi: 10.1021/bi00541a003. [DOI] [PubMed] [Google Scholar]
  27. Rosenberg S., Lawrence J., Stracher A. Effect of various extraction solutions and thrombin activation on the composition of the platelet cytoskeleton. Cell Motil. 1982;2(4):317–332. doi: 10.1002/cm.970020402. [DOI] [PubMed] [Google Scholar]
  28. Small J. V. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J Cell Biol. 1981 Dec;91(3 Pt 1):695–705. doi: 10.1083/jcb.91.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stenberg P. E., Shuman M. A., Levine S. P., Bainton D. F. Redistribution of alpha-granules and their contents in thrombin-stimulated platelets. J Cell Biol. 1984 Feb;98(2):748–760. doi: 10.1083/jcb.98.2.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. White J. G., Estensen R. D. Degranulation of discoid platelets. Am J Pathol. 1972 Aug;68(2):289–302. [PMC free article] [PubMed] [Google Scholar]
  31. White J. G. Exocytosis of secretory organelles from blood platelets incubated with cationic polypeptides. Am J Pathol. 1972 Oct;69(1):41–54. [PMC free article] [PubMed] [Google Scholar]
  32. Zucker M. B. The functioning of blood platelets. Sci Am. 1980 Jun;242(6):86–103. doi: 10.1038/scientificamerican0680-86. [DOI] [PubMed] [Google Scholar]

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